Obesity is characterized by low-grade chronic inflammation. As an acute-phase reactant to inflammation and infection, C-reactive protein (CRP) has been found to be the strongest factor associated with obesity. Here we show that chronic elevation of human CRP at baseline level causes the obesity. The obesity phenotype is confirmed by whole-body magnetic resonance imaging (MRI), in which the total fat mass is 6- to 9- fold higher in the CRP rats than the control rats. Univariate linear regression analysis showed different growth rates between the CRP rats and the control rats, and that the difference appears around 11 weeks old, indicating that they developed adult-onset obesity. We also found that chronic elevation of CRP can prime molecular changes broadly in the innate immune system, energy expenditure systems, thyroid hormones, apolipoproteins, and gut flora. Our data established a causal role of CRP elevation in the development of adult-onset obesity. Chronic inflammation is a player in the progressive process of obesity development .The transgene contained the human CRP gene , and the mouse albumin promoter (from +22 to −300 bp) and enhancer (from −12.171 to −9.469 kb) . PurifieThe expression of the human CRP transgene and rat endogenous gene was measured by quantitative real-time PCR (qRTPCR) , ELISA  and WestTotal RNA samples were extracted from the liver with the RNeasy Mini Kits (Qiagen) after homogenization with FastPrep Lysing Matrix Tubes D . Reverse transcription was performed with 0.5 μg of total RNA using a SuperScriptIII First-Strand Synthesis kit (Invitrogen). The real-time PCR was performed with a Faststart DNA Master SYBR Green kit (Roche) on a Light Cycler.Whole blood was withdrawn from the tail vein of the animals. The serum was obtained by a centrifugation at 13,000 rpm at 4°C for 10 min. ELISA was performed to measure the protein levels of human CRP expression using a human CRP specific ELISA kit  and rat CRP expression using a rat-specific ELISA kit . The serum samples were diluted 1:6,000 fold for the human CRP ELISA and 1:20,000 for the rat CRP ELISA, and the ELISA experiments were carried out following the manufacturers' protocols.For the Western blot experiments, the serum was diluted at 1:1000, and then loaded onto 4–12% Bis-Tris polyacrylamide gels. It was transferred to PVDF membranes, blocked, and then incubated with anti-human CRP antibody . Membranes were then incubated with the secondary antibody , washed three times, and then incubated with substrate solution  for 5 min and exposed to X-ray film.2 (cm2).Longitudinal growth in all animals was followed up from the age of 5 to 40-weeks old. Body weight, and body length (nose-anus length) were measured weekly in the morning throughout the follow-up period. The body weight and body length were used to determine the body mass index as follows: BMI = body weight (g)/lengthMRI had become one of the ultimate methods to directly measure the fat distribution in animal models. In this study, we scanned the transgenic rats and the control rats on a 3T MRI scanner  with the Siemens 4-channel flex coil in the Emory Yerkes National Primate Research Center primate center. The structural T1-weighted images of the whole body were acquired using a spin-echo sequence with the acquisition parameters: TR = 2,520 ms, TE = 15 ms, FOV = 340 × 340 mm, data matrix = 384 × 384, slice thickness = 2 mm, 50 slices, 6 averages. Total scan duration was 24 min for each rat. Raw data from the MRI scans were processed using the MRIcron software toolkit to covert to analytical format and create, view, and analyze the three-dimensional images. The quantitative measurements of fat in all rats were carried out on the sagittal slice crossing the renal hilum with the Image J software.Food consumption was measured daily in the morning at the same time (9:00 to 10:00 am) continuously from 8 to 12-weeks old for 1 month. Every morning, sufficient food was added after being weighed. The next morning, the residual food was weighed again. The difference was the consumptive food by rats in the same cage. Based on food and caloric intake, the following nutritional parameter was calculated: Energy intake (kJ/day) = mean food consumption × dietary metabolic energy.The tissues of fat and spleen were collected. The organs were weighted, and gross appearance was examined immediately. The collected tissues were kept in 10% buffered formalin, embedded, and cut into 5-micron-thicksections. The tissue sections were mounted on slides and stained with hematoxylin & eosin (H&E) or Oil Red O. The spleen sections were further stained with anti-CD3 antibody  to demonstrate the T lymphocytes, and with anti-CD20 antibody  to show the B lymphocytes. The secondary antibodies were sc-2039 for CD3 and sc-2042 for CD20. The images were taken by the Picture FrameTM Application 2.3. The number of splenic white pulps was counted at 4×magnification in 10 areas of the spleen sections from either transgenic rats or control rats. The results of Immunohistochemistry were quantified by Image-Pro Plus software (version 6.0). The pathological experiments were performed by the Histo-Pathology Core of Atlanta VA Medical Center, and pathological interpretations were performed by the Emory Department of Pathology.The rats were fasted overnight for 15 h and blood was withdrawn from caudal vein. The prepared serum was either assayed immediately or stored in aliquots at −80°C for later use, avoiding repeated freeze/thaw cycles. The lipid profiles were measured by the Emory Lipid Research Laboratory of Atlanta Clinical and Translational Science Institute (ACTSI). Briefly, triglyceride (TG) was measured in serum by enzymatic method with the Triglyceride Quantification Kit ; total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured with the HDL and LDL/VLDL Cholesterol Assay Elisa Kit .n = 5; line 519, n = 6; controls, n = 4). All blood samples were sent to Antech Diagnostics  for the hematological analysis. Siemens Advia 120 Multispecies Hematology System was used in the hematological analysis.We carried out a complete blood count (CBC) to evaluate the composition and concentration of the cellular components of blood in the transgenic rats and control rats. Blood was withdrawn from the 26-week-old transgenic rats and the control rats  and PathCards via the GeneCards portal.Blood was withdrawn from 16-to 17-week-old rats, stored at 4°C overnight, and then centrifuged at 3,000 rpm at 4°C for 15 min. Highly abundant plasma proteins, such as albumin, IgG, and transferrin, were depleted from samples using a Multiple Affinity Removal Column . The samples were then concentrated, and total protein concentrations were measured with a BCA Protein Assay Kit . About 300 μg of depleted serum proteins were dissolved in 200 mM triethyl ammonium bicarbonate (TEAB). Following reduction and alkylation, serum proteins were digested into peptides with trypsin  at 37°C overnight. Peptides were then labeled with TMT6 (126 - 131 Da) . TMT126, 127, and 128 were used to label control (non-transgenic) samples in triplicates, and TMT129, 130, and 131 were used to label samples from the transgenic rats in triplicates. Labeled peptides were combined and fractionated with strong cation exchange (SCX) column (Thermo Scientific) in high-performance liquid chromatography (HPLC) (Shimazdu) and analyzed in an TQ-Orbitrap XL instrument (Thermo-Fisher Scientific) coupled to an Ultimate 3000 Dionex nanoflow LC system . Peptides were identified according to high mass resolution, and TMT6 reporter ion quantification was recognized with high energy collision dissociation (HCD). Proteins were searched for each identified peptide with Mascot . The ratios of TMT6 reporter ion abundances in MS/MS spectra generated by HCD (up to six reporter ions ranging from m/z 126.12 to m/z 131.14) were used to calculate fold changes of proteins between the control groups and the transgenic groups. From the raw proteomic results, all proteins with ELISA was used to measure blood levels of a series of hormones using a series of specific commercial kits. ELISA experiments were outsourced to the ACTSI/Emory Cardiovascular Specialty Laboratories . Briefly, serum samples were collected from transgenic lines (488 and 519) and their non-transgenic littermates (controls). Insulin and glucagon were measured by ELISA using the commercial kits. The sources of commercial ELISA kits are as follows: Insulin (Mercodia), Glucagon (R&D Systems), triiodothyronine (T3) , total thyroxine (T4) , thyroid-stimulating hormone (TSH) (ALPCO), Leptin , Adiponectin (R&D Systems), TNFα (R&D Systems), IL6 (R&D Systems), and Complement C3 .http://www.arb-silva.de/), which has taxonomic categories predicted to the genus level.Total microbial DNA was extracted from fecal samples using Qiagen stool DNA extraction kit (Cat. No. 51504) following the manufacturer's protocol and then sequenced by the 16S RNA sequencing on an Illumina HiSeq2500 sequencer. Sequences were grouped into operational taxonomic units (OTUs) using the clustering program UCLUST and assigned OTUs into taxonomic categories using the Ribosomal Database Silva_111 16S rRNA database. PCoA was calculated with the QIIME pipeline. The Ribosomal Database Program classifier was used to assign taxonomic category to all OTUs at confidence threshold of 0.97. The Ribosomal Database Program classifier uses Silva_111 16S rRNA database  using the Bonferroni post-hoc test for multiple comparisons. The linear regression was used to investigate the relationship between the body weight and the age of rats. In our experiment, the body weight was treated as the corresponding variable, and the age (week) was employed as the explanatory variable in the statistical analysis. Different datasets were combined based on the timeline (week is used as the time unit) of up to 40 weeks. After the combination, there were a total of three groups of rats, line 519, line 488, and non-transgenic littermates of SD rats included. The significance levels were set at 0.05 for all tests. The SAS statistical package 9.3  was used for all data managements and analyses.All data were expressed as mean ± standard deviation; the statistical significance was analyzed by Student's We noticed that the CRP transgenic rats were big and strong, so we started to monitor the longitudinal growth of body weight and nose–anus length during the period from 5-weeks old to 40-weeks old. We found that the human CRP transgenic rats were larger in gross appearance, with higher body weight than their non-transgenic littermates . UnivariThe fat volume was measured by a standard T1-weighted (T1w) whole-body magnetic resonance imaging (MRI) to validate that these transgenic rats had more fat. As shown by the 2D images on the sagittal slices, the axial slices, the coronal slices , the conp = 3.1E-48) higher than the control rats (51.7 ± 10.9)  was significantly ( ± 10.9) . The adiFood intake was followed up among rats from 8 to 12-weeks old. We found no significant difference between transgenic rats and control rats . We alsop < 0.001) (CRP transgenic rats displayed a noteworthy splenic remodeling. The number of white pulps (the reservoir of T-cells and B-cells) in the CRP transgenic rats was about 2.5-fold higher than control rats (< 0.001) . The sta< 0.001) .p < 0.05) between transgenic rats and control rats, and the difference on the neutrophil count reached statistical significance only in one transgenic line (p < 0.05)  .In concert with the splenic remodeling, we also observed striking differences in the subcomponent levels of complete system between the CRP transgenic rats and control rats. The subcomponents of complement system in the classical pathway, including C1qA, C1qB, C1r, C1s, C2, C4b, and C4, were all upregulated by 2-to 4-fold, and the membrane attack subcomponent C9 was down-regulated . ComplemOur results showed remarkable differences between the CRP transgenic rats and control rats on the protein levels on a series of the key proteins that play critical roles in the control of immune response and energy expenditure. Corticosteroid-binding globulin  was substantially upregulated by 2.2-fold in the CRP rats compared with the control rats . Two othp = 0.028; line 519, 4.72 ± 0.34 μg/dl, p = 0.024) than the control rats (3.87 ± 0.64 μg/dl). Their T3 levels  seemed to be slightly lower than those of the control rats (0.63 ± 0.08 ng/ml) but did not reach statistical significance. On the plasma concentration of TSH, there was no statistical significance between transgenic line 488 and the control rats, but transgenic line 519 had significantly lower TSH compared with the control rats .We measured the serum levels of T3 and its prohormone, T4, as well as TSH produced from the pituitary gland . Those CP = 0.003 for line-519 vs. control; 0.014 for line-488 vs. control)  were about 2-fold higher in the CRP rats than the levels in the control rats; the concentration of apolipoprotein ApoB-100 was decreased by 1.6-fold . The tricontrol) . We obsep < 0.05) than the non-transgenic group .Our sequencing results showed that Firmicutes and Bacteroidetes were the two predominant phyla among the bacterial groups, contributing 57.7 and 32.5% of gut flora in the transgenic rats, and 55.5 and 32.9% in the non-transgenic littermates. Proteobacteria and Actinobacteria constituted the next dominant phyla at 3.2 and 0.3% in transgenic rats, and 3.3 and 0.4% in the non-transgenic littermates . The abuic group . At the Akkermansia was significantly lower (p < 0.05) in the transgenic rats compared with the non-transgenic littermates (Butyrivibrio (11.6-fold) and Peptococcus (1.8-fold) were significantly higher  than that in non-transgenic littermates  on the serum levels between transgenic rats and non-transgenic littermates . Among tThis study demonstrates that the long-term chronic CRP elevation will lead to adult-onset obesity, which is based on results of the CRP transgenic rat model with low levels of human CRP expression. As shown by whole-body MRI scanning, fat mass of transgenic rats is 6-to 9-fold higher than the fat mass of control rats, among which 12–23% is subcutaneous fat, 77–88% is intra-abdominal fat, and 42–63% is omental fat. The adipocytes in the CRP rats (mean diameter = 75.8 microns) is three-times bigger than the fat cells in the control rats (mean diameter = 51.7 micron). The increase of adiposity might be an essential factor in expression of CRP and chronic inflammation leading to obesity, which needs to be further explored.CRP has been known as an inflammation marker, but our results show that CRP is more than a marker; it can play an active role to provoke systematically the innate system. Chronic CRP elevation regulates the levels of complement components in the classical pathway ; it moduBesides the innate immune system, CRP also remarkably modulates the key proteins in the regulation of energy expenditure and immune response . Among tAkkermansia muciniphila is a well-known “weight-loss bacteria” (Everard et al., A. muciniphila (iniphila . ThinkinIn human, CRP is consistently the strongest factor associated with overweight and obesity as revealed by large epidemiological studies (Timpson et al., In summary, long-term CRP elevation can lead to adult-onset obesity. CRP can also provoke a broad adjustment in the innate immune system and energy expenditure system. CRP may be a new therapeutic target for obesity and its complications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All animal experiments were performed with the approval of the Animal Care Committee of Morehouse School of Medicine, conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.Conceptualization: QS. Data curation: YC, SY, YD, QiW, and QL. Formal analysis: XZ, WL, and YM. Investigation: QL, QingwW, WX, YM, QiW, QingW, WL, XZ, SY, JZ, GW, and LZ. Methodology: QL, DE, MB, JC, and LM. Resources: QingwW, QS, QL, and BW. Supervision: QS and QL. Writing-original draft: QL, QiW, YM, WX, and QingwW. Writing-review and editing: QS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, silver microspheres (SMS) were introduced into cotton stalk porous-carbon (CSP) to prepare silver microsphere doping porous-carbon (SMS-CSP), and then SMS-CSP was used as the matrix of polyethylene glycol (PEG) to synthesize shape-stable phase change material of PEG/SMS-CSP. It was found that the introduction of SMS into CSP could not only greatly improve the loading capacity of the porous-carbon for PEG, but also could increase the thermal conductivity of PEG/SMS-CSP. Additionally, the method of introducing SMS into porous-carbon had the advantages of environmental protection and simple operation. Moreover, the raw material of cotton stalk is a kind of agricultural waste, which has the merits of wide source, low price and easy to obtain. Furthermore, in the preparation of cotton stalk porous-carbon, with the increase of pyrolysis temperature the thermal conductivity of PEG/SMS-CSP could be enhanced significantly. The mechanism about the enhancement of thermal conductivity was clarified, which could provide more basic theory for the study about the thermal conductivity of shape-stable phase change materials (ss-PCMs) based on porous-carbon. Latent heat energy storage is to store or release energy by absorbing or releasing heat in the process of phase change by using phase change materials (PCMs). The reusability, high energy storage density and high heat recovery rate of the PCMs had helped to attract much attention2. PCMs are widely used in varied fields, such as power peak-load shifting, building energy saving, industrial waste heat utilization and solar energy utilization3. PCMs could be classified as organic PCMs and inorganic PCMs on the basis of chemical structure. Among them, organic PCMs mainly include polyethylene glycol (PEG), paraffin, higher fatty acids, polyolefin, etc., which possess a series of advantages such as good fixed molding, less prone to phase separation, and small corrosion. However, the organic PCMs still have the disadvantages of seepage and low thermal conductivity, which hinder their practical application.On the basis of rapid economic development, the imbalance between the energy consumption and supply makes people aware of the importance of renewable energy. Energy storage technology is of great significance for solving energy shortage problems. Up to now, energy storage technologies could be classified according to energy storage medium, which could be divided into mechanical energy storage, latent heat energy storage, electrical energy storage and chemical energy storage4. Many porous materials, such as mesoporous silica5, diatomaceous earth6, expanded graphite7, graphene8, aerogel9, metal foam10, metal organic skeleton11 and biochar12 were used as the support materials. Biochar is a carbon-rich material that could be produced from a variety of organic waste materials such as agricultural waste and municipal sewage sludge by pyrolysis of biomass under anoxic or anaerobic conditions. Biochar is receiving more and more attention due to its unique characteristics such as high carbon content, large specific surface area and stable structure14. Wu et al.15 utilized biochar as a soil improvement agent to increase the available phosphorus content in the saline-alkali soil and reduce the leaching of phosphorus in the soil.Fixing the organic PCMs in porous materials to synthesize shape-stable phase change materials (ss-PCMs) is an effective way to solve the above problems14 utilized organic-type PCMs as core material and rice husk biochar as porous supporting material to design bio-based ss-PCMs, and the obtained ss-PCMs exhibited high thermal stability. Chen et al.16 prepared ss-PCMs with biochar as the matrix and polyethylene glycol as the core material by vacuum impregnation method, and the results showed that the ss-PCMs had favorable thermal stability and good thermal reliability. However, the thermal conductivity of ss-PCMs prepared using biochar as the carrier still needed to be improved. Recent studies found that adding high thermal conductive nanoparticles to ss-PCMs is an effective method to enhance their thermal conductivities. Oya et al.17. utilized graphite to fix erythritol for the preparation of high thermal conductive ss-PCMs by adding nickel particles. Cheng et al.18 designed ss-PCMs of tetradecanol/expanded perlite, and the thermal conductivity of tetradecanol/expanded perlite was improved by adding Cu powder. However, there are still some defects in the preparation of ss-PCMs with improved thermal conductivity by adding high thermal conductive nanoparticles, such as high cost, complex process and easy to pollute the environment.Additionally, the leakage of organic PCMs could be avoided by using biochar as carrier to prepare ss-PCMs. For example, Jeon et al.19, and in that study, mesoporous carbon was used to adsorb copper ions firstly, and then copper ions were reduced to copper microspheres by in-situ reduction at high temperature. Then mesoporous carbon doping with copper microspheres was obtained, which was used as the support of organic PCM to prepare ss-PCMs with improved heat conduction performance. This method has the advantages of simple operation, environment-friendly and low cost. Additionally, the copper microspheres synthesized by in-situ reduction method could be firmly combined on the surface of mesoporous carbon, which could prevent the deposition of copper microspheres in the phase transformation process of ss-PCMs and increase the thermal conductive stability. However, there are still some disadvantages about this thermal conductivity enhancement method, such as low loading content of core material and relatively small increase range of the thermal conductivity.In order to solve the above problems, our research group developed an in-situ reduction method for the preparation of ss-PCMs with improved thermal conductivity based on carbon material21, and then silver microspheres were synthesized by in-situ reduction strategy on the surface of CSP. The ss-PCMs (PEG/SMS-CSP) was prepared by using PEG as core material and silver microsphere doping cotton stalk porous-carbon (SMS-CSP) as the carrier. The thermal properties of PEG/SMS-CSP were studied in detail.Aim to further improve the loading capacity and thermal conductivity of ss-PCMs that was prepared by using in-situ reduction strategy, in this study, the cotton stalk, which is a kind of agricultural waste and has the advantages of wide source, low price, easy to obtain, high carbon content, etc., was used as the raw material to prepare cotton stalk porous-carbon (CSP)In this paper, it was found that the introduction of SMS into CSP could not only greatly improve the loading capacity of the porous-carbon for PEG, but also could increase the thermal conductivity of the ss-PCMs of PEG/SMS-CSP. Moreover, the method of introducing silver microspheres into porous-carbon had the advantages of environmental protection and simple operation. More importantly, to the best of our knowledge, there was no study report about the effect of different pyrolysis temperature on the properties of ss-PCMs used porous-carbon as the supporter, and in this study, it was found that with the increase of pyrolysis temperature of cotton stalk porous-carbon, the thermal conductivity of PEG/SMS-CSP could be enhanced significantly, and the enhancement mechanism was clarified.The cotton stalks were obtained from Chuzhou, Anhui, China. Silver nitrate was obtained from Shanghai Zhanyun Chemical Co, Ltd, and ethanol was obtained from Chemical Reagent Sinopharm Holding Co, Ltd. Polyethylene glycol (Mn = 4000) was obtained from Aladdin Reagent Co, Ltd.The schematic diagram for the synthesization of PEG/SMS-CSP is shown in Fig. The PEG/SMS-CSP with different PEG contents was prepared by vacuum impregnation method. PEG solutions with different concentrations were synthesized by adding a certain amount of PEG  into 15 ml anhydrous ethanol. After that a certain amount of SMS-CSP  were added into the above solutions. The above suspension was agitated in vacuum condition for 1 h, and then continuously stirred in a constant temperature water bath for 1 h at 45 °C. Finally, the black solid product was dried at 50 °C for 12 h, and three kinds of ss-PCMs were prepared, which were named as 50% PEG/SMS-CSP, 60% PEG/SMS-CSP and 70% PEG/SMS-CSP. For comparison, the 50% PEG/CSP and 60% PEG/CSP without SMS were prepared by using the same synthesis procedure as for 50% PEG/SMS-CSP and 60% PEG/SMS-CSP.2 atmosphere from 50 to 500 °C with heating rate of 10 °C/min. The specific surface area and pore volumes of the SMS-CSP and CSP was evaluated through Brunauer–Emmett–Teller (BET) method .The enthalpy and phase transition temperature of PEG, CSP and PEG/SMS-CSP samples were accurately measured by utilizing differential scanning calorimetry , which were carried out in the temperature range of 0 °C to 100 °C by using high purity nitrogen as the protective gas, and the heating and cooling rate was 10 °C/min. X-ray photoelectron spectroscopy  was used to record the spectra of CSP and SMS-CSP. The transient plane source method  was used to measure the thermal conductivities of PEG and PEG/SMS-CSP.The morphologies of CSP, SMS-CSP and PEG/SMS-CSP were investigated by scanning electron microscopy . Spectroscopic analysis was performed by using Fourier transform infrared spectrometer , and the crystalloid phase of the samples was tested by wide-angle X-ray diffraction .Thermo gravimetric analysis was utilized to test the thermal stability of PEG and PEG/SMS-CSP in N22. In order to directly and clearly observe the distribution characteristics of silver element, the element surface distribution of 70% PEG/SMS-CSP was tested, and Fig. The SEM images of CSP, SMS-CSP and 70% PEG/SMS-CSP are shown in Fig. 2/g, and its pore volume was 0.12 cm3/g. It could be seen from Fig. 3/g, and its specific surface area was 509.04 m2/g. It could be seen that the specific surface area of CSP was 344.23 m2/g, which was much less than that of SMS-CSP. Compared with CSP, the higher micro/mesopore proportions were observed for SMS-CSP, which had a significant impact on PCM encapsulation. During the phase transition process, a large micro/mesopore proportion could prevent the organic PCMs from leakage because of the strong capillary force23. Therefore, introducing silver microspheres into CSP could increase its specific surface area, which could enhance the loading capacity of SMS-CSP for organic PCMs.The pore size distribution diagram, nitrogen adsorption and desorption isotherms of CSP and SMS-CSP are shown in Fig. −1 was attributed to the stretching vibration of –OH24. The absorption peak at 3415 cm−1 was attributed to the stretching vibration peak of –OH19. The absorbing peak at 2877 cm−1 was because of the symmetrical stretching vibration of CH226. The adsorption band at 1122 cm−1 was because of the stretching vibration of C–O, and the absorption peak at 932 cm–1 was the in-plane deformation vibration of C–O16. For the 70% PEG/SMS-CSP, all of the characteristic absorption peaks of PEG and SMS-CSP could be found in the spectra of 70% PEG/SMS-CSP, and no new peak appeared. Therefore, it was only physical interaction in the preparation of 70% PEG/SMS-CSP, which showed that the SMS-CSP developed in this study was ideal carriers for PEG immobilization, and the as-prepared ss-PCMs of 70% PEG/SMS-CSP had favorable compatibility and chemical stability.The chemical stability and chemical compatibility of the samples could be studied by the FT-IR spectroscopy. Figure 23. For the pattern of SMS-CSP and 70% PEG/SMS-CSP, two different diffraction peaks were found at 38.10° and 44.30°, which represented the crystal surface of the silver monomer. This result indicated that silver microspheres were successfully introduced into CSP. Two typical peaks in PEG pattern appeared at 19.20° and 23.30°, and these two peaks could also be found in the 70% PEG/SMS-CSP pattern, which had the same shape, indicating that PEG was fixed in SMS-CSP successfully. Additionally, even when PEG was fixed in SMS-CSP, PEG could still maintain excellent crystalline properties and its crystal structure was not damaged. However, the diffraction peak of 70% PEG/SMS-CSP was significantly smaller than that of pristine PEG, which meant that the crystallinity of 70% PEG/SMS-CSP was lower than that of pristine PEG. It might be due to that the pore structure of SMS-CSP limited the crystallinity of PEG molecules in 70% PEG/SMS-CSP, and this result could be attributed to the Van der Waals force and capillary force between SMS-CSP and PEG molecules. In addition, no new peak appeared in the XRD pattern of 70% PEG/SMS-CSP, which indicated that there was no chemical reaction in the preparation process of 70% PEG/SMS-CSP.XRD could be used to measure the chemical compatibility and crystallization properties of the samples. The XRD patterns of CSP, SMS-CSP, 70% PEG/SMS-CSP and PEG are shown in Fig. 3/2 (374.28 eV) and Ag 3d5/2 (368.23 eV), indicating that the successful introduction of SMS into CSP.The elemental composition of CSP and SMS-CSP was determined by XPS. The XPS curves of CSP and SMS-CSP were shown in Fig. 23. According to the results, the maximum PEG loading amount in PEG/CSP was 50%, and however, the maximum PEG loading amount in PEG/SMS-CSP could reach 70%. This result could be attributed to that when SMS was introduced into CSP, the surface area of SMS-CSP was increased obviously compared to that of CSP, which could also be verified by the study results of BET tests. Therefore, the adsorption capacity of SMS-CSP was much larger than that of CSP, and the loading capacity of SMS-CSP was improved significantly.Leakage experiments, which were used to determine and compare the shape stability of pure PEG, 50% PEG/CSP, 60% PEG/CSP, 60% PEG/SMS-CSP, 70% PEG/SMS-CSP and 80% PEG/SMS-CSP, were carried out to check whether they would leak by putting them into a baking oven at 65 °C for 10 min, and the results are exhibited in Fig. 25. However, according to the study results, the 70% PEG/SMS-CSP still had high-temperature resistance and excellent thermal stability.Figure 26. Table f), freezing latent heat (ΔHf), melting temperature (Tm), and melting latent heat (ΔHm). The study results proved that the SMS-CSP hardly had influence on the phase transformation temperature of PEG. For instance, the freezing and melting temperatures of pristine PEG were 62.80 °C and 34.00 °C, respectively, and for the 70% PEG/SMS-CSP, the values were 60.90 °C and 33.20 °C, and the similar study results were widely mentioned in the literatures, such as GO/PEG27 and SiO2/PEG28 composite PCMs. In Table 29.The DSC curves of PEG, 50% PEG/SMS-CSP, 60% PEG/SMS-CSP and 70% PEG/SMS-CSP are shown in Fig. 31. For a weak interaction between porous materials and organic PCMs, the melting point would decline, while a strong interaction would result in an increase of phase change temperature. Additionally, the PEG molecules might interact with the porous carbon through hydrogen bonding and capillary force, which led to lower melting point30. The difference value of Tf and Tm is expressed as the supercooling temperature (ΔT)31. As can be seen from Fig. The melting points of 50% PEG/SMS-CSP, 60% PEG/SMS-CSP, and 70% PEG/SMS-CSP were decreased slightly in comparison with that of pure PEG. The changes in melting points were representation of the strength of interactions between the supporting materials and PEGIn order to study the thermal reliability of PEG/SMS-CSP, the DSC tests for 50% PEG/SMS-CSP, 60% PEG/SMS-CSP and 70% PEG/SMS-CSP before and after 100 thermal cycles were carried out, and the results are shown in Figs. Figure 3 in the preparation process of PEG/SMS-CSP possessed an obvious effect on the thermal conductivity of the ss-PCMs, and Fig. 3 concentration. When the concentration of AgNO3 was 200 mg/L, it could be seen that the thermal conductivity of PEG/CSP was 0.42 W/(m K), and when the concentration of AgNO3 was 400 mg/L, the thermal conductivity of 70% PEG/SMS-CSP increased gently. When the AgNO3 concentration was 600 mg/L, the thermal conductivity of 70% PEG/SMS-CSP increased suddenly, which might be due to the fact that when the AgNO3 concentration was 400 mg/L, a small number of silver microspheres were obtained by in-situ reduction at 1300 ºC, and the silver microspheres could not contact with each other, resulting in the inhibition of electron transfer between silver microspheres particles, and leading to the decrease of the thermal conductivity of 70% PEG/SMS-CSP. When the AgNO3 concentration was 600 mg/L, more silver microspheres could be obtained, and the spherical structure of silver microspheres played a "bridging" role between PEG and porous-carbon, which provided a favorable heat flow channel and promoted the formation of thermal network and led to the rapid increase of thermal conductivity of the 70% PEG/SMS-CSP. When AgNO3 concentration was 800 mg/L, the thermal conductivity of 70% PEG/SMS-CSP could reach 0.78 W/(m K), which was increased by 300% compared to that of the pure PEG. It could be found that when the concentration of AgNO3 increased from 600 to 800 mg/L, the increment of the thermal conductivity of 70% PEG/SMS-CSP was slight. The reason was because when the concentration of AgNO3 was 600 mg/L, the adsorption amount of silver ions by cotton stalk porous-carbon was nearly saturated. Hence, when the concentration of AgNO3 increased to be 800 mg/L, the increment of the adsorption quantity of silver ions by CSP was small, and therefore, after in-situ reduction the amount of silver microspheres on the surface of CSP increased a little, resulting in a slight increase for the thermal conductivity of 70% PEG/SMS-CSP while the concentration of AgNO3 increased from 600 to 800 mg/L.The concentration of AgNO32. Therefore, the thermal conductivity of 70% PEG/SMS-CSP was enhanced with the increase of the pyrolysis temperature. Table Figure According to the above study results, it was demonstrated that the non-uniformly dispersed spherical silver microspheres on the surface of CSP formed a thermal conductive path in the matrix, which improved the thermal conductivity of PEG/SMS-CSP dramatically. In addition, we found that the thermal conductivity of PEG/SMS-CSP could be improved by adjusting the structure of porous-carbon through changing the pyrolysis temperature, which provided more means for the thermal conductivity enhancement of ss-PCMs.3 concentration were optimized. The results showed that the maximum content of PEG in PEG/SMS-CSP was 70%, and with the increase of pyrolysis temperature and AgNO3 concentration, the thermal conductivity of PEG/SMS-CSP could be enhanced. The thermal conductivity of 70% PEG/SMS-CSP could reach 0.78 W/(m K), which was increased by 300% compared to the pure PEG, and the phase change enthalpy of 70% PEG/SMS-CSP was 122.9 J/g. Furthermore, the PEG/SMS-CSP possessed remarkable thermal stability and thermal reliability, and therefore, the PEG/SMS-CSP developed in this study could be applied as an ideal thermal energy storage material.In this study, a novel shape-stable phase change material of PEG/SMS-CSP was developed using silver microsphere doping porous-carbon as the matrix. The preparation conditions such as PEG content, pyrolysis temperature and AgNO


To improve interfacial adhesion between glass fiber (GF) and poly(ethylene-grafted-styrene)-based cation exchange membranes (CEM), GF was modified by four coupling agents: [3-(Methacryloxy)propyl] trimethoxy silane (3-MPS), 1,6-bis (trimethoxysilyl) hexane , Poly (PP-g-MA) and Triethoxyvinylsilane (TES). The results indicated the addition of modified GF increased tensile strength, tensile modulus, storage modulus and interfacial adhesion of GF/CEM composite but degraded the strains. The composite with 3-MPS modified GF obtained superior mechanical properties and interfacial adhesion, whereas the modified effect of TES was inconspicuous. The addition of unmodified GF even had negative effects on GF/CEM mechanical properties. The field emission scanning electron microscopes (FE-SEM) showed that the GF treated by 3-MPS and PP-g-MA have better compatibility with the CEM matrix than 1,6 bis and TES-treated GF. The Fourier-transform infrared spectroscopy (FT-IR) verified that the strengthening effects from modified GF were attributed to the formation of Si-O-Si and Si-O-C bonds. The additions of modified GF in CEM positively influence water uptake ability but negatively influence ion exchange capacity (IEC). This research provided a way of strengthening GF/CEM composite and pointed out which functional groups included in coupling agents could be useful to GF-reinforced composite. Over recent decades, there has been renewed interest in developing high-performance ion exchange membranes, which play important roles in various industrial applications, such as fuel cell, desalination and wastewater treatment ,2,3,4,5.According to the connecting way of charged groups on the matrix, ion exchange membranes can be classified into homogeneous and heterogeneous membranes . HomogenGlass fiber (GF)-reinforced polymer composite gradually became a competitive structural material due to the excellent mechanical properties. Three factors affect the fiber-reinforced composite’s mechanical properties: intrinsic properties of matrix materials, the strength of interfacial chemical bonding between the fillers and matrix and the load transfer efficiency of interphase . A largeRecently, many methods have been studied to improve the interfacial adhesion and construct chemical bonds between the fibers and matrix. The styrene/2-ethylhexylacrylate/divinylbenzene solid foams was reported to be reinforced by the sonicated silica particle up to 3%. The sonication-treated silica particle considerably enhanced the composites’ Young’s modulus and crush strength . Zhang eThe idea of improving interfacial adhesion between the fiber and polymeric matrix by modifying the fiber surface can also be introduced to GF and ion exchange membrane composite. In Křivčík’s work, the polypropylene and GF were used as a low-cost alternative to common woven fabric in ion exchange membrane . He confThe objective of this article is to investigate the modifying effects of different coupling agents on GF, which were used to strengthen poly(ethylene-co-styrene)-based cation exchange membrane. The mechanical properties, morphology characterization and FT-IR of GF-reinforced poly(ethylene-grafted-styrene)-based cation exchange membrane (CEM) composites were studied in this paper. To assure the mechanical property enhancement is not at the cost of other membrane properties, the ion exchange capacity and water uptake ability were also investigated in this paper.1,6 bis was supplied by Gelest Inc; 3-MPS and PP-g-MA  were supplied by Sigma Aldrich Co, Ltd. St. Louis, MO, USA); TES was purchased from TCI Co, Ltd. ; low density polyethylene films  were supplied by Uline Co. Ltd. . GF was supplied by Fibre Glast Developments Corporation , with a diameter of sixteen microns and average length of two hundred and thirty microns. Styrene (99.9% purity), divinylbenzene , Luperox A98 , 1,2-dichloroethane, chlorosulfonic acid and polystyrene-block-poly-(ethylene-ran-butylene)-block-polystyrene were purchased from Sigma Aldrich Co, Ltd. . Toluene, xylene and hydrochloric acid  were supplied by Carolina Chemical Co, Ltd. . All solvents were used as received.The coupling agents were hydrolyzed in ethanol alcohol solution, adjusting pH to 3.5 using diluted hydrochloric acid. The concentration of coupling agents in ethanol solution was 2%. After 1 h hydrolyzation, GF was immersed in the different coupling agent solutions for 1 h at 100 °C. Then, the glass fibers were dried at room temperature for 24 h. The chemical structure and hydrolyzation mechanism of these four coupling agents are illustrated in When the effect of the coupling agent’s concentration on the mechanical properties of composite was investigated, the concentration in ethanol solution varied to 1%, 3%, 4% and 5%, respectively.LDPE films were rinsed with acetone and dried at 50 °C for 6 h to remove moisture. Then, 2 g LDPE was dissolved in the mixture composed of 20 mL toluene and 2 mL xylene at 90 °C. After LDPE was completely dissolved, the styrene, DVB and benzoyl peroxide were added to the above mixture in the nitrogen atmosphere. The reaction was kept at 90 °C for 6 h. The mass ratio between PE and styrene was 1:1.5. The DVB and benzoyl peroxide were added at a weight ratio of 3% and 0.5% to styrene. When the reaction was completed, the copolymer was stirred with the GF at 200 °C for 20 min. The formulation of composite with untreated GF, treated GF and GF treated by different coupling agent concentrations is summarized in Sulfonation of GF/PE-g-PS membrane composite was carried out by immersing it in the chlorosulfonic acid solution  at 0 °C for 2 h. After sulfonation, the resulting membrane composite was repeatedly rinsed with distilled water to remove residual chlorosulfonic acid, and the cation exchange membrane composite was obtained after drying in an oven at 50 °C overnight.−1. Tensile strength was tested at the loading rate of 0.5000 N·min−1 until 5.0000 N.Mechanical properties. The evaluation of mechanical properties to GF/CEM composites includes dynamic mechanical analysis (DMA) and tensile strength tests. These tests can be obtained from the DMA Q 800 machine . The DMA provides storage modulus and loss factor, being monitored from 25 to 150 °C, at a heating rate of 3.00 °C·minMorphology characterization. The microstructure, compatibility of GF and the matrix membrane and phase separation of composites were characterized by FE-SEM analysis. The Hitachi S-4700 FE-SEM  is equipped with an Oxford energy dispersive X-ray spectroscopy (EDS) microprobe .Fourier-transform infrared spectroscopy (FT-IR) analysis. The functional groups of the matrix membranes and final composite were investigated by FT-IR. A PerkinElmer Spectrum One FT-IR spectrometer  with a universal attenuated total reflection accessory was used to record the infrared spectrum.+ form. Then, the membranes were washed with distilled water to remove the excess sulfuric acid. The resulting membranes were dried in the oven at 60 °C and then stirred with 50 mL 0.5 M NaCl solution overnight. Due to the replacement of H+ on the membrane by sodium cation from the solution, the solution becomes acidic. The concentration of H+ was determined by pH measurement and titration using a diluted solution . The IEC values were calculated using the Equation (1):KOH is the volume of KOH used in the titration, and Wdry is the dry weight of the membrane in g. CKOH is the molarity(mol/L) of KOH used in the experiment for the titration.Ion exchange capacity (IEC). The IEC of CEM was determined by the titration method. The dried membranes were cut into small pieces and stirred with 1.0 M sulfuric acid solution overnight to make sure sulfonate groups are in Hwet). The wet membrane was dried until the water evaporated completely and weighed again (Wdry). The water uptake can be determined by Equation (2).Water uptake. The water uptake experiments were performed by measuring the weight differences between dried membranes and fully hydrated membranes. The membranes were cut into small pieces and immersed in deionized water at 25 °C with a designed duration. After that, the surface moisture was removed, and mass was weighed :The effects of various coupling agents on mechanical properties of GF/CEM composite are illustrated in Not only the types of coupling agent affect composites’ mechanical strength but also the coupling agent’s concentrations. Since the 3-MPS showed a superior modification effect on GF, the 3-MPS modified GF was chosen to investigate the effect of coupling agent concentration on mechanical properties. The dynamic mechanical property can be evaluated by storage modulus and loss modulus. The storage modulus reflects the ability of materials storing energy elastically, representing the elastic portion. The loss modulus reflects the ability of materials dissipating stress through heat, representing the viscous portion. The loss modulus to storage modulus ratio in a viscoelastic material is defined as the tan delta, which provides a measure of damping in the material.The influences of various coupling agents on storage modulus as a function of temperature are presented in c and tan δm represent the loss factors for the composite and matrix, respectively. Vf represents the volume fraction of glass fibers. The parameter A is introduced to measure the adhesive effects between the fillers and matrix; A is defined as Equation (5):The loss factor (tan δ)–temperature curves in i is ignored, then Equation (4) can be rewritten as Equation (6):Assuming Hence, the parameter A can be rewritten as Luis Ibrarra’s formula (Equation (7)):c and tanδm. The A values of GF/CEM composites with GF treated by 3-MPS and PP-g-MA were relatively lower than others, indicating higher glass fiber–matrix adhesive effects obtained at their interface. 3-MPS and PP-g-MA were also demonstrated have better modification effects on improving the adhesive capacity of the interface. The negative value of A in the blank sample represents the addition of raw GF impaired interface adhesion. The applied force could not dissipate from the matrix to GF.The A is an inverse proportion to the interfacial adhesion. The effects of 3-MPS’s concentration on DMA as a function of temperature are shown in The surface morphologies of CEM composites with untreated GF and GF treated by four coupling agents were investigated by FE-SEM , which were from the condensation between -OH in GF and the Si-OH groups in coupling agents. A new appearing peak at 1065 cm−1 in the spectrum of sample 6 was attributed to asymmetric vibrations of C-O-Si bonds, which originated from the condensation between -OH in GF and C-OH in PP-g-MA. Both emerging peaks were the evidence of a forming chemical bonding between GF and coupling agents. The peaks intensities around 3660 cm−1 in spectrum 3, 4, 5 and 6 amplified due to -Si-OH bonds from hydrolyzed 1,6 bis, 3-MPS, TES and PP-g-MA. In addition, the peaks around 2850–3000 cm−1 and 1400 cm−1 were attributed to C-H in methyl groups and C-H in aliphatic, respectively. The intermolecular hydrogen bonding among the GF, coupling agents and PE-g-PS matrix was characterized by these C-H stretching vibration. The C-H stretching vibration regions of 3,4,5,6 spectra presented on the right graph were shifted to the higher wavenumbers compared to the C-H spectra in pure PS-g-PE. This change in the C-H absorptions indicated the specific effect of hydrogen bonding between the matrix and coupling agents. Based on the above analysis, the possible reaction mechanism of GF, coupling agents and PE-g-PS copolymer is deduced as The FT-IR spectra of pure PE-g-PS copolymer and that with untreated GF and treated GF are shown in −1 (Si-O3CH2−) became sharpest when 3-MPS concentration reached 2%. Increasing 3-MPS concentration weakened corresponding peak intensities. The less sharp characteristic peaks indicate that lower amounts of Si-O-Si bonds generated between GF and coupling agents. The -C-H peaks attributed to methyl groups and aliphatic had the same trends as Si-O-Si peaks, indicating the more PE-g-PS could be attached on GF when GF was treated by 2% 3-MPS hydrolyzed solution. These results demonstrate that the 2% 3-MPS concentration has a superior modified effect on GF.Since the mechanical properties are strongly related to the coupling agent’s concentration, the effects of concentration on formation of chemical bonding were investigated by the FT-IR spectrum. −1. The IEC of CEM composites with treated GF decreased slightly, 2–6% lower than CEMs without GF. The negative influence of adding GF on IEC value was in an acceptable range. However, the addition of untreated GF decreased the IEC value tremendously due to the vast emerging holes caused by GF insertion, which intercepted the ion transportation channels. The coupling agents could fill those vacancies, making the GF combine with the matrix tightly. The cations transported through aqueous membranes under the “diffusion mechanism”. Hydrated cation (H3O+) diffused through the aqueous medium in response to the electrochemical difference in this mechanism. The water connected cation (H+ (H2O)x) carried one or more water molecules transported through the membrane. Under this condition, the cations could bypass the obstacles and conduct through branch ionic groups of PE-g-PS membrane when encountering GF.The concern of adding fillers is that it may hinder ion transport and decrease ion exchange capacity. The result in −1 NaCl for 2 h. Water uptake is a vital property since it positively correlates to IEC. However, excessive water uptake would result in a swollen membrane. Thus, the enhanced water uptakes of CEMs with 3-MPS, PP-g-MA and TES-treated GF lead to a higher IEC but increase the risk of unstable dimension.Adding GF into CEMs also affects water uptake and swelling rate due to the hydrophilic nature of GF. This research provided information about interfacial adhesion and mechanical property improvement effects of four coupling agents on GF/CEM composite. The addition of modified GF enhanced the tensile strength and tensile modulus of GF/CEM composites but could not make composites suffer too much elongation. The interfacial adhesion between the matrix and GF, reflected by tan delta and the adhesive effect parameter, were improved with the addition of coupling agents due to the formations of Si-O-Si bonds between GF/coupling agent, and hydrogen bonding among the GF, coupling agents and matrix. The 3-MPS treated-GF showed satisfactory modification consequences, brought the most robust interface adhesion to the composite and led to the highest tensile strength. However, with increasing 3-MPS concentration to treat GF, the mechanical properties and interfacial adhesion worsened. The unmodified GF negatively influenced the composite’s mechanical properties due to the destruction of the membrane’s integrity and formation of micro-holes around the GF. The water uptake values increased after adding modified GF into CEMs. However, the IEC of GF/CEM composite decreased slightly. The swelling rate for the unmodified GF/CEM composite is relatively high due to the absence of chemical bonding between the GF and composite matrix.This research provided a way of strengthening GF/CEM composite and pointed out which functional groups included in coupling agents could be useful to GF-reinforced composite. Future studies of GF-reinforced ion exchange membrane could use these conclusions as references. They might also consider other surface modifiers with similar characteristic structures or functional groups as effective coupling agents for GF-reinforced CEM composite.


Measuring health-related quality of life (HRQOL) in patients with chronic low back pain (LBP) is crucial to monitor and improve the patients’ health status through effective rehabilitation. While the 12-item short-form health survey (SF-12) was developed as a shorter alternative to the 36-item short-form health survey for assessing HRQOL in large-scale studies, to date, no cross-culturally adapted and validated Hausa version exists. This study aimed to translate and cross-culturally adapt the SF-12 into Hausa language, and test its psychometric properties in mixed urban and rural Nigerian populations with chronic LBP.The Hausa version of the SF-12 was developed following the guidelines of the International Quality of Life Assessment project. Fifteen patients with chronic LBP recruited from urban and rural communities of Nigeria pre-tested the Hausa SF-12. A consecutive sample of 200 patients with chronic LBP recruited from urban and rural clinics of Nigeria completed the instrument, among which 100 respondents re-tested the instrument after two weeks. Factorial structure and invariance were assessed using confirmatory factor analysis (CFA) and multi-group CFA respectively. Multi-trait scaling analysis  and known-groups validity were performed to assess construct validity. Composite reliability (CR), internal consistency (Cronbach’s α), intraclass correlation coefficients (ICC), and Bland–Altman plots were computed to assess reliability.After the CFA of the original conceptual SF-12 model, 2 redundant items were removed and 4 error terms were allowed to covary, thus providing adequate fit to the sample. The refined model demonstrated good fit and evidence of factorial invariance in three demographic groups . Convergent  and divergent  validity were satisfactory. Known-groups comparison showed that the instrument discriminated well for those who differed in age (p < 0.05) but in gender and habitation (p > 0.05). The physical component summary and the mental component summary demonstrated acceptable CR (0.69 and 0.79 respectively), internal consistency (α = 0.73 and 0.78 respectively), test-rest reliability (ICC = 0.79 and 0.85 respectively), and good agreement between test-retest values.The Hausa SF-12 was successfully developed and showed evidence of factorial invariance across age, gender, and habitation. The instrument demonstrated satisfactory construct validity, internal consistency, and test-retest reliability. However, stronger psychometric properties need to be established in general population and other patients groups in future studies. The instrument can be used clinically and for research in Hausa-speaking patients with chronic LBP. Low back pain (LBP) constitutes a significant problem of the contemporary society as it affects people all age groups . It is nChronic LBP is an important clinical and public health problem as sufferers may continue to experience pain and functional disability which often interfere with daily life activities and subsequently reduce quality of life . The assThe medical outcomes study 36-item short-form health survey (SF-36) is perhaps the most widely used instrument to assess perceived health status. Since its development, it has been used as a generic instrument to evaluate or monitor HRQOL in the general population and people with different chronic illnesses including LBP –18. HoweThe SF-12 consists of 12 items taken from the eight subscales of the SF-36. Similar to the SF-36, it assesses two global health constructs viz the physical component summary (PCS) and the mental component summary (MCS) . The SF-The adaptation of health status measures for use in other than the source language is essential since it does not only permit the collection of valid and reliable data but also minimize the exclusion of subjects who cannot speak the source language ,28. HoweThis study aimed to translate and cross-culturally adapt the SF-12 into Hausa language, and test its psychometric properties in mixed urban and rural Nigerian populations with chronic LBP.This study was approved by the Health Research Ethics Committee, Ministry of Health Kano State (Ref: MOH/Off/797/T.I./651). Written informed consent was obtained from all participants prior to participating in the study.Translation, cross-cultural adaptation and cross-sectional study of psychometric properties.www.orthotoolkit.com/sf-12/) to compute the PCS-12 and MCS-12 scores.The SF-12 consists of 12-items and 8 subdomains: physical functioning (PF), role-physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role-emotional (RE), and mental health (MH). The subscales PF, RP, BP, and GH forms the physical component summary (PCS-12) scores whereas the subscales VT, SF, RE, and MH forms the mental component summary (MCS-12) scores. Each item of the questionnaire has response categories which vary from 2- to 6-point scales and raw scores for items ranging from 1 to 6. The raw scores are summated and linearly transformed into 0–100 scale  with a hWritten permission to translate the SF-12 health survey into Hausa language was obtained from the original developer. The cross-cultural adaptation followed the guidelines recommended by the International Quality of Life Assessment (IQOLA) project .Two bilingual (English and Hausa) translators independently forward-translated the English SF-12 into Hausa. The first translator was a professional linguist and unaware of the concepts of the questionnaire. The second translator was a clinical physiotherapist with ample experience in questionnaire translation and aware of the concepts being examined. The aim of reaching conceptual equivalence with the original source version rather than literal equivalence while reflecting the lay language used in Hausa culture regardless of age and educational level was emphasized to both the translators. This stage led to the production of two forward Hausa SF-12 versions which were then reconciled and synthesized into one version following discussion and consensus among the forward translators with coordination of the primary author. The synthesized version was then translated back into English by two, independent professional translators who had no medical background and access to the original version of the questionnaire. This ensured that the translated questionnaire was reflecting the meaning in the original questionnaire .To evaluate face validity, an expert review committee consisting of all the translators, the primary author and an academic physiotherapist with proficiency in methodology met and produced a pre-final version of the Hausa SF-12 after reaching consensus. The pre-final version was then pilot-tested in 15 patients with chronic LBP recruited from both urban and rural communities to assess comprehensibility and applicability. Upon completion of the questionnaire, cognitive debriefing  was carried out by asking the participants to comments on the questionnaire items and their perceived meaning of chosen responses. The primary author with the consultation of the translators and methodologist then reviewed the questionnaire for problematic items, responses, statements, phrases, and words in terms of clarity and acceptability. This ensured that the original meaning was not lost or altered while reaching cultural equivalence. Finally, a professional translator independently proofread the final Hausa SF-12 translation for any minor errors that may have been missed during the translation and cultural adaptation process. This led to the production of the final version of the Hausa SF-12 see .The procedure used throughout this section has been used in the cross-cultural adaptation of other Hausa self-report measures as described elsewhere .The “Quality criteria for measurement properties of health status questionnaires” suggest that a sample size of ≥ 50 would be sufficient for reliability, construct validity, and ceiling/floor effects analyses whereas 4–10 subjects per variable (Rules-of-thumb) would be sufficient for factorial structure analysis . Based oThe participants were consecutive LBP patients presenting to the out-patient clinics of Murtala Muhammad Specialist Hospital, Dawakin-Kudu General Hospital, Wudil General Hospital, and Kura General Hospital all in Kano State, Northwestern Nigeria, between February and May 2018. Urban patients were recruited from the specialist hospital while rural patients were recruited from the general hospitals. Both urban and rural patients were recruited to have broader applicability of the instrument in both urban and rural areas, as well as across all levels of literacy or illiteracy. Participants were included if they were both sexes, aged 18–70 years, suffering from LBP for 12 weeks or greater, and fluent in Hausa language. They were excluded if their LBP was due to serious spine pathology , cognitive impairment or impaired capacity to be interviewed, and pregnancy.Training of assessors. Physiotherapists in the respective hospitals received training on interviewer-administration of measures as many patients in Nigeria especially rural dwellers are not literate (ability to read and write in Hausa). This was deemed necessary to minimize survey error even though majority of the physiotherapists are familiar with the questionnaire. All the physiotherapists were staff of Hospital Management Board Kano State, Nigeria, with clinical experience between two to five years. The therapists received a one-day training session based on verbal pretesting of measures. The session included face-to-face and group-based training coordinated by the primary researcher in a classroom.Data collection. Information on demographic characteristics  and clinical data (duration of pain) were obtained and recorded for each participant. The Hausa SF-12 was interviewer-administered. However, literate participants’ were self-administered where necessary. The questionnaire was re-administered at an interval of approximately 14 days after the first measurement to minimize participants recalling previous answers.All statistical analyses were performed using IBM SPSS for Windows version 24.0  at an alpha level of 0.05 except for confirmatory factor analysis (CFA) which was performed with IBM AMOS software, version 26.0 for Windows. Descriptive statistics of mean, standard deviation (SD), frequencies, and percentages were used to summarize the data. Visual  and statistical methods (Kolmogorov-Smirnov and Shapiro-Wilk’s test) were used to test the normality of the data.Floor or ceiling effects are considered to exist if more than 15% of respondents scored the minimum or maximum possible score . PotentiFactor structure of the Hausa SF-12 was investigated by first performing exploratory factor analysis (EFA) using principal component analysis with varimax rotation to verify the original conceptual SF-12 model. It was hypothesized that a two-factor model (reflecting PCS-12 and MCS-12) would be obtained with eigenvalues greater than 1 ,34. ConfFactorial invariance or measurement invariance was investigated by performing multi-group CFA across age , gender (men and women), and habitation  groups. We categorized age into two groups only as further categorization would lead to severely unbalanced groups  which might affect the results . Factorir) were interpreted as being strong (> 0.6), moderate (0.3–0.6), and weak/low (< 0.3) [Construct validity was investigated by assessing convergent, divergent, and known-groups validity. Convergent and divergent validity were assessed using multi-trait scaling analysis with the use of Pearson’s correlation coefficients . Pearson’s correlation coefficients ( (< 0.3) .r > 0.4) than other component [r < 0.4) [r < 0.4) since they measure a different latent concept [For convergent validity, it was expected that item scores would correlate higher with own hypothesized component  was assessed by comparing mean scores of scales and components by age, gender, and habitation using one-way analysis of variance (ANOVA) or independent t-test. Effect sizes were interpreted according to Cohen’s d as either trivial (< 0.2), small (≥ 0.2 and < 0.5), moderate (≥ 0.5 and < 0.8) or large (≥ 0.8) . We hypoInternal consistency for the PCS-12 and MCS-12 was assessed using Cronbach’s alpha (α). A Cronbach-α value of ≥ 0.70 is generally regarded as acceptable .Test-retest reliability of the PCS-12 and MCS-12 was assessed by calculating intraclass correlation coefficient (ICC) for agreement using a two-way random effects ANOVA model (which assumes that measurement errors could arise from either raters or subjects). Confidence intervals (CI) were also computed for the ICC. A coefficient ≥ 0.70 was considered adequate for test-retest reliability . AdditioThe translation of the Hausa SF-12 was easy as there were no major translation problems encountered except for items 2 and 11. In item 2, the phrase “pushing a vacuum cleaner and bowling” was modified to “lifting a dustbin and archery”. In the Hausa culture, pushing a vacuum cleaner is not familiar, hence the phrase “lifting a dustbin” was used. Similarly, bowling which refers to a target sport and recreational activity that involves rolling or throwing a heavy ball towards a target is not commonly practiced in Hausa culture. In contrast, archery which involves a skill of using a bow to shoot arrows is commonly practiced in the Hausa culture and can be an alternative to bowling. Furthermore, in item 11, the phrase “depressed and heart-broken” was used in place of the phrase “down-hearted and blue” as this phrase has no equivalence in the Hausa culture. The translators ensured that the original meaning is not lost or altered while attaining cultural equivalence between the original source version and the Hausa version. Results of the pilot testing suggest that all the items were clear and comprehensive.All the respondents completed the instrument signifying 100% response rate. There were 123 (61.5%) males and 77 (38.5%) females. Their mean age was 45.5±14.5 years with majority of them living in rural areas (60%). Slightly over half of the respondents were Hausa non-literates (55.5%) and self-employed farmers and traders (56.0%). The demographic characteristics of the respondents are fully shown in All the 200 respondents completed the Hausa SF-12 without missing values. The mean score for the PCS-12 was 34.5 (SD = 6.94), and 38.9 (SD = 10.1) for the MCS-12. Floor effects were found in items 2–7  whereas ceiling effects were found in items 1 , 5 , 7 , and 10  .The two-factor conceptual model of the SF-12 was confirmed explaining 49.7% of the total variance following the EFA. Factor one included items 6, 7, 9, 10, 11, and 12, which reflect MCS-12 while factor two included items 1, 2, 3, 4, 5, and 8 which reflect PCS-12. The item-factor loadings (λ) for the PCS-12 ranged .41–.75 while that of the MCS-12 ranged .52–.73. The CFA for the original conceptual SF-12 model and refined model fitted to the Hausa sample of patients with chronic LBP is presented in The results of the multi-group CFA across age, gender, and habitation are presented in n = 200) of the Hausa SF-12. Regarding convergent validity, items pertaining to physical health correlated higher with the PCS-12 except for item 4 (RP1) (r < 0.4) whereas items pertaining to mental health correlated more with the MCS-12, all , thus confirming the hypothesized item component correlations. For divergent validity, items belonging to the PCS-12 had the lowest correlation with the MCS-12 whereas items belonging to the MCS-12 had the lowest correlation with the PCS-12 except for item 5 (RP2) and 8 (BP) (r > 0.4), all , thus confirming the hypothesized item component correlations. The results also showed that the PCS-12 and MCS-12 were weakly correlated (r = 0.18) to each other, thus indicating discriminant validity as hypothesized  with small to moderate effect size . The youn = 200) as measured by Cronbach-α if item deleted was 0.73 for the PCS-12 and 0.78 for the MCS-12.Internal consistency (n = 100) as measured by ICC was 0.79 (95% CI: 0.69–0.86) for the PCS-12 and 0.85 (95% CI: 0.77–0.89) for the MCS-12. The Bland–Altman analysis showed a mean difference of –0.96 and 0.55 for PCS-12 and MCS-12 respectively. The limits of agreement for PCS-12 were –11.387 to 9.467 and –11.739 to 12.839 for MCS-12. The results show minimal systematic bias , unlike in the reversed version (SF-12v2) where the item response has been extended from 2 to 5 which gives more response categories .Though the AVE values obtained for the PCS-12 and MCS-12 were smaller than the acceptable value of ≥ 0.5 , howeverTo the best of our knowledge, this is the first study to examine the factorial or measurement invariance of the SF-12 in population with chronic LBP. Interestingly, the proposed refined model exhibited a good fit in the demographic groups according to age, gender, and habitation. Factorial invariance of the Hausa SF-12 was fully supported evidence by the adequate model fit statistics and indices in terms of configural, metric and scalar invariance analyses. These findings suggest the ability of the Hausa SF-12 to perform similarly well among younger adults and adults, men and women, as well as urban and rural populations with chronic LBP. Our findings are in concordance with that reported by Galenkamp et al  who founa priori hypotheses were confirmed for the convergent  and divergent  validity. Convergent validity was demonstrated by the higher correlations of items with own hypothesized component whereas divergent validity was demonstrated by the lower correlations of items with component less in common. However, item 5 (RP2) and 8 (BP) which supposed to correlate higher with the PCS-12, also had a relatively high correlation with the MCS-12. This is somewhat similar to the findings obtained for the original English SF-12 version reported by Ware et al [Results of the construct validity of the Hausa SF-12 were very encouraging as the re et al  where thre et al , Tunisiare et al , and Morre et al , howeverKnown-groups validity of the Hausa SF-12 was supported in terms of its ability to differentiate between subgroups of respondents who differed in age but in gender and habitation. Older respondents were found to exhibit poor health in the scores RP, BP, GH, VT, and MH scales, as well as PCS-12, compared to younger respondents. These findings are consistent with the results of previous studies on general population ,34. ThouInternal consistency for the PCS-12 (0.78) and MCS-12 (0.79) lies within the recommended Cronbach-α range of 0.70–0.95 , thus inThis study is not without potential limitations which should be considered when interpreting the results. Firstly, though our translation process followed the IQOLA protocol, few optional steps such as rating of the difficulty and quality of the forward and backward translations were skipped due to lack of funding and limited resources. Secondly, the study included only patients with chronic LBP; thus, the study results may not be generalized. It should be noted that clinimetric properties of a measure are influenced by population characteristics and so can change in different population groups ,67. FurtThe results of this study suggest that the Hausa SF-12 was successfully developed and showed evidence of factorial invariance across age, gender, and habitation. The construct validity, internal consistency, and test-retest reliability were satisfactory. However, stronger psychometric properties need to be established in general population and other patients in future studies. The instrument proved to be useful for clinical and research purposes in Hausa-speaking patients with chronic LBP. It may also support the uptake of multicentric and multinational studies such as the global health initiatives which usually involve concurrent research activities in culturally and linguistically diverse countries.S1 Appendix(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


The proteasome inhibitor bortezomib is currently commonly used for the treatment of multiple myeloma (MM). MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in messenger RNA silencing and post-transcriptional regulation of gene expression. In MM, the expression of several miRNAs is markedly dysregulated suggesting their role in MM pathogenesis and drug resistance. The aim of our study was to assess miRNA expression patterns in the serum of MM patients treated with bortezomib. We have identified 21 serum miRNAs differentially expressed in patients refractory to bortezomib-based chemotherapy. A miRNAs-based prediction model was developed to assess the probability of refractoriness to bortezomib. Our findings, indicating the differential expression of miRNAs between bortezomib-refractory and bortezomib-sensitive patients, suggest that these circulating miRNAs may play an important role in personalized treatment of MM patients.Bortezomib is the first-in-class proteasome inhibitor, commonly used in the treatment of multiple myeloma (MM). The mechanisms underlying acquired bortezomib resistance in MM are poorly understood. Several cell-free miRNAs have been found to be aberrantly regulated in MM patients. The aim of this pilot study was to identify a blood-based miRNA signature that predicts bortezomib-based therapy efficacy in MM patients. Thirty MM patients treated with bortezomib-based regimens were studied, including 19 with refractory disease and 11 who were bortezomib sensitive. Serum miRNA expression patterns were identified with miRCURY LNA miRNA miRNome PCR Panels I+II (Exiqon/Qiagen). Univariate analysis found a total of 21 miRNAs to be differentially expressed in patients with MM according to bortezomib sensitivity. Multivariate logistic regression was created and allowed us to discriminate refractory from sensitive patients with a very high AUC of 0.95 (95%CI: 0.84–1.00); sensitivity, specificity and accuracy were estimated as 0.95, 0.91, and 0.93. The model used expression of 3 miRNAs: miR-215-5p, miR-181a-5p and miR-376c-3p. This study is the first to demonstrate that serum expression of several miRNAs differs between patients who are bortezomib refractory and those who are sensitive which may prove useful in studies aimed at overcoming drug resistance in MM treatment. Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM) microenvironment ,2. It isMultiple myeloma often results in bone lesions, hypercalcemia, infections, anemia and production of monoclonal immunoglobulin. The interaction of MM cells with host factors, and the BM microenvironment plays a key role in the molecular evolution of the disease and the generation of treatment-resistant cells; it also influences disease progression with the development of relapsed or refractory disease ,5. The tMicroRNAs (miRNAs) are small, non-coding RNAs only 19 to 25 bases in length that are secreted into the circulation and influence the regulation of gene expression in physiological or pathological conditions ,11,12. MIn patients with MM, miRNAs seem to be involved in drug resistance ,19,20,21predominant paraprotein detected was IgG type (60.0%) followed by light chain myeloma (23.3%). Most of the patients received a bortezomib, cyclophosphamide and dexamethasone (VCD) regimen; however, two patients received VMP , and one received VTD . Nineteen patients were refractory and 11 sensitive to bortezomib-based therapies. Overall, no statistically significant differences were observed between the refractory and sensitive group regarding the studied clinical variables , miR-376c-3p  and miR-181a-5p  were associated with refractoriness to bortezomib treatment . Internal validation of the selected panel model using bootstrap optimism corrected AUC suggests that the panel offers high discriminatory power when applied to independent samples .The Hosmer-Lemeshow test for goodness of fit indicated good fit of the model  have not been investigated in MM patients yet. However, the results of several studies indicate the involvement of these molecules in the biology of other neoplasms. MiR-143-3p was identified as the key specific mediator of KIAA1429 expression in osteosarcoma cells . In addiMiR-16-2-3p demonstrates strong tumor suppressive and antimetastatic properties in osteosarcoma and other neoplasms . SeveralThe serum levels of miR-744-5p, miR-151a-3p, miR-376a-3p, miR-191-5p, miR-181a-5p, miR-766-3p, miR-30c-5p, miR-130a-3p, miR-409-3p, miR-1224-3p and miR-328-3p, which were lower in the refractory group than the sensitive patients . MiR-181Although no previous studies have confirmed that other miRNAs are down-regulated in bortezomib refractory MM, they are known to be involved in the development of various cancers, and some of them play an important role in cancer progression. Overexpression of miR-744-5p was found to inhibit the proliferation, migration, and invasion of cancer cells in epithelial ovarian cancer, non-small cell lung cancer and in nasopharyngeal carcinoma ,60. MiR-Some of the miRNAs identified in our study as being differentially expressed in bortezomib refractory and sensitive MM patients are known to be potentially important for prediction of response to treatment in other cancers. MiR-151a-3p could predict the response to azacitidine treatment in myelodysplastic syndromes when tested in combination with miR-423-5p, miR-126-3p miR-125a-5p, and miR-199a-3p . Using CFinally, combining miRNAs with anti-MM drugs may improve treatment efficacy and prevent drug resistance among patients with MM ,76,77,78Elsewhere, miR-221/222 overcame melphalan resistance and triggered apoptosis of MM cells in vitro: Gulla et al. demonstrated that inhibition of miR-221/222 via LNA-i-miR-221 triggers cell apoptosis in vitro and restores drug sensitivity in melphalan-refractory MM cells . In addiPSMB5, CDK5 and CYP1A1, in MM cells [Our findings demonstrated that serum expression of several miRNAs differs between patients who are refractory to bortezomib and those who are sensitive. However, it is important to examine the roles of these miRNAs in the regulation of recently-identified genes associated with bortezomib resistance, such as MM cells ,82,83. IMM cells . HoweverA total of 30 MM patients who were admitted to the Department of Hematology, Copernicus Memorial Hospital, Lodz, Poland were included in the study. The group of patients included 18 men and 12 women with a mean age of 64 years . Their demographic, clinical and laboratory details are shown in Response to treatment and relapse/progression events were classified according to the International Myeloma Working Group (IMWG) ,86. SensThe study was conducted according to good clinical and laboratory practice rules and the principles of the Declaration of Helsinki. All procedures were approved by the local ethical committee . Each patient signed the informed consent for all examinations and procedures.g for 10 min. Serum samples were stored at −80 °C. for further use.Venous blood samples were collected from MM patients before treatment with bortezomib based regimens. Peripheral blood was collected in serum separating tubes and processed within 2 h of collection by centrifugation at 2400× Isolation of total RNA including miRNA was performed from serum using the miRNeasy Serum/Plasma Advanced Kit  according to the manufacturer’s protocol. Samples of 2 μL of the final eluate was used as the sample input. To allow for normalization of sample-to-sample variation in RNA isolation, prior to purification, each serum sample was spiked with UniSp2 (2 fmol), UniSp4 (0.02 fmol), UniSp5 (0.00002 fmol), each at a different concentration in 100-fold increments and UniSp6 (0.075 fmol) and cel-miR-39-3p (0.001 fmol) as a positive control for cDNA synthesis. RNA quality was determined with Agilent High Sensitivity RNA ScreenTape using 2200 TapeStation . Directly after isolation RNA was subjected to the reverse transcription process.MmiRNA profiling was performed by miRCURY LNA RT Kit (Qiagen) and used to prepare the reverse transcription reaction according to the manufacturer’s recommendations. Real-time PCR was performed on a LightCycler480 Real Time PCR System  using and miRCURY LNA miRNA miRNome PCR Panel I and II. This array enables accurate quantitation of 752 human microRNAs.Mature miRNAs were polyadenylated by poly(A) polymerase and reverse transcribed into cDNA using oligo-dT primers. Polyadenylation and reverse transcription were performed in parallel in the same tube. The oligo-dT primers have a 3′ degenerate anchor that allows amplification of mature miRNA in the real-time PCR step. MiRCURY LNA Reverse Transcription Kit was purchased from Qiagen and used to synthesize cDNA according to the guidelines provided by the manufacturer. Two µL of isolate was added to the reaction tube to make up a final volume of 10 μL reaction mix. The reaction took place at 42 °C for 60 min and were then inactivated at 95 °C for 5 min in thermal cycler.The expression of 752 miRNAs were determined in all patients using miRCURY LNA miRNome Human PCR Panel I and II (Qiagen). Real-time PCR was performed on LightCycler480 II (Roche) instrument. Three µL of cDNA template (diluted 1:60) was used in each PCR reaction. The reaction was performed at 95 °C for 2 min, followed by 45 amplification cycles at 95 °C for 10 s and 56 °C for 1 min. Fluorescence was measured after each cycle. Relative quantification of mRNA was determined by a comparative Ct method.miRNA selection, batch correction was performed using ComBat [miRNAs were filtered for miRNAs present in 100% of both groups, leaving 155 miRNAs to further analysis. After g ComBat . The stag ComBat ,91. ThisNormalized ΔCt = mean Ct of miR-23b-3p, miR-151a-5p and miR-152-3p—Ct miRNA.This approach results in higher values for higher miRNA expression enabling easy biomarker interpretation. Normalized ΔCt for all samples and with class assignments were provided as Nominal variables were expressed as percentages and analyzed using the Chi-square test with appropriate corrections if needed: the Yates correction for continuity or Fisher’s exact test. The normality of the distribution of continuous variables was verified with the Shapiro-Wilk test. For continuous variables, the difference between two groups was evaluated using a two-sided independent Student’s t test when the data was normally distributed, and with the Mann-Whitney U test when the assumption of normality was not met or if the variable was ordinal. Continuous variables were presented as mean ± standard deviation (SD) or medians with 25% to 75% values according to the data distribution. For visual representation of multidimensional data, principal component analysis (PCA) and unsupervised hierarchical clustering were performed.For a more comprehensive analysis, a logistic regression model was generated that used the refractoriness to a bortezomib-based regimen as outcome and miRNAs as predictors. The variables were preselected for the development of the classification model using Student’s t-test. All miRNA with FDR < 0.2 were chosen as candidate variables in this step. We estimated both a univariate model for each of the selected miRNA and a multivariate model that included all selected predictors. Forward stepwise and backward stepwise selection approaches were used to restrict the model to the most predictive miRNAs. The model with the lowest AIC (The Akaike information criterion) value was chosen as the final model. The predictive power of the final model was evaluated by receiver operating characteristics (ROC) and area under the curve (AUC) analysis to determine the ability of the biomarker panel to accurately predict refractoriness to the bortezomib-based treatment regimen. The goodness of fit of the model was tested with the Hosmer-Lemeshow statistic, where high p values indicate a good fit.Bootstrap samples were used to test for possible overfitting by determining optimism values on calibration measures. The bootstrap analysis was performed on 1000 different samples of the same sample size drawn with replacements from the original sample. Optimism, which is a measurement of internal model validation that refers to the absolute magnitude of bias, equals the difference between respective statistics of the bootstrap sample (bootstrap performance) and the bootstrap model in the original sample (test performance) . Bias-comiRNAselector R package [p values.All statistical analyses were conducted using Statistica Version 13.1  and R programming language (version 4.0.2). Most of the analysis utilized our  package  P valuesIn conclusion, our data demonstrate differential serum miRNA expression between patients with MM who are sensitive to bortezomib-based therapies and those who are not. To our knowledge, this is the first study to demonstrate such differences between these two groups of patients. These results indicate a promising therapeutic target for coping with drug resistance in MM treatment. In addition, we describe the development of a multivariate regression model for predicting refractoriness to bortezomib-based treatment in MM patients. Our findings provide evidence that combined expression levels of miR-215-5p, miR-376c-3p and miR-181a-5p can be used to predict the response to bortezomib treatment. Internal validation of the selected panel suggests that the discriminatory power of the panel will be high when applied to independent samples. In addition, miRNAs are promising therapeutic target for coping with drug resistance in MM treatment. However, the clinical and biological importance of these findings need further investigation.